1. Field of the Invention
The present invention relates to an online electrophoresis apparatus for determining base sequences by electrophoresing DNA fragment samples and detecting fluorescence from the samples during the electrophoresis. Primers or terminators of the DNA fragment samples are labeled by the Sanger's method. More specifically, the present invention relates to an electrophoresis apparatus called a multi-capillary DNA sequencer for simultaneously electrophoresing a plurality of samples by employing a plurality of capillary columns charged with electrophoresis gels.
2. Description of the Background Art
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for determining the base sequences of DNAs such as human genomes, which have long base sequences. Accordingly, a multi-capillary DNA sequencer that has a plurality of capillary columns charged with gels is proposed in place of those employing flat plate type slab gels. As compared with the slab gels, the capillary columns can readily handle, inject and electrophorese samples at a high speed and make highly sensitive detections. If a high voltage is applied in slab gels, bands are spread due to the influence of Joulean heat, or temperature gradients are caused. Conversely, capillary columns have no such problems but make highly sensitive detections with minimal spreading of bands under high-speed electrophoresis with the application of a high voltage.
A treatment by the Sanger's method results in the formation of four types of DNA fragment samples having ends consisting of A (adenine), G (guanine), T (thymine) and C (cytosine). In order to increase the throughput, it is necessary to electrophorese the samples in a plurality of capillary columns. When a set of samples of end bases are electrophoresed in different capillary columns, however, differences between the electrophoresis speeds of the capillary columns result in errors of base sequence determination. Therefore, a set of samples containing DNA fragments of four types of end bases must be injected into each capillary column. In this case, each capillary column labels the four types of DNA fragments of different end bases with at least two types of fluorescent materials in order to distinguish the fragments from each other.
A method has been proposed (refer to Anal. Chem. 1994, 66, pp. 1021-1026 (reference 1)) employing four types of fluorophores FAM, JOE, TAMRA and ROX as labels for different DNA fragments in order to distinguish the DNA fragments of different end bases. A method has also been proposed (refer to Anal. Chem. 1992, 64, pp. 2149-2154 (reference 2)) employing two types of fluorophores FAM and JOE in different ratios for distinguishing four types of DNA fragments from each other through coding. Both cases provide examples of samples containing four types of DNA fragments of different end bases being injected into each capillary column in order to increase the throughput of a multi-capillary DNA sequencer. DNA fragments of different end bases can be distinguished from each other by multicolor fluorescent labels and a plurality of samples can be simultaneously electrophoresed in a plurality of capillary columns.
Samples are simultaneously electrophoresed in a plurality of capillary columns, so that an optical system detects fluorescence generated from the samples. An example of such an optical system introduces an excitation light beam from the side of an array of the capillary columns and detects the generated fluorescence with a CCD (charge-coupled device) camera which is positioned perpendicularly along the capillary column array (refer to reference 1). In order to eliminate background signals based on scattering on the surfaces of the capillary columns, the electrophoresed samples are rendered to form sheath flows on positions irradiated with the excitation beam in the exterior of the capillary columns.
While the beam intensity is less damped in the method of reference 1 due to the formation of the sheath flows, it is difficult to position the capillary columns for implementing the sheath flow states, and multiplication is hard to attain.
Another exemplary optical system condenses excitation light in order to irradiate a single capillary column in an excitation optical system, for detecting fluorescence from samples in the capillary column by a photoreceiving optical system. Both excitation and photoreceiving optical systems are fixed in such a way as to perpendicularly scan an array of a plurality of capillary columns, thereby successively detecting fluorescence from samples in the capillary columns (refer to reference 2).
In the method of reference 2, however, the capillary array is mechanically moved in order to be scanned, resulting in the capillary columns being twisted so that the number thereof tends to be restricted.
In each of these methods, the number of capillary columns for simultaneously performing electrophoresis is limited and results in a restriction of the number of simultaneously analyzable samples. There is also room for improvement in the throughput
In the case of detecting fluorescence from a capillary array with an image pickup device, such as a CCD or a CID (charge-injection device), the number of detectable capillary columns is limited due to limitation of the pixel number of the image pickup device. Furthermore, an image pickup device, such as a CCD or a CID, has a lower sensitivity than a photomultiplier.
In the case of employing a number of capillary columns, it is difficult to inject different samples into the respective columns. Furthermore, ends of the capillary columns must be dipped in samples contained in the sample injection vessels so that the samples are injected into the same by voltage application or the like, which are then transferred into reservoirs storing buffer solutions for electrophoresis. Thus, a great deal of time is required for injecting the samples and starting electrophoresis thereof, and it is therefore convenient if these operations can be automatized.
While capillary electrophoresis apparatuses employing only a single capillary column are able to maintain at a constant temperature within that column, this is not the case with multi-capillary electrophoresis apparatuses. Therefore, electrophoresis speeds are dispersed due to temperature changes in electrophoresis, or the spaces between detected bases fluctuate (compression), and result in errors in base sequence determination.
A multi-capillary base sequence determining apparatus obtains four types of detection signals for respective fluorescence wavelengths, which are set to correspond with the end bases. The apparatus displays four types of signal waveforms for the respective end bases on the screen of a display unit, and displays what bases are currently being detected in the respective capillary columns during electrophoresis. If the detected waveforms are displayed as such in the case of making electrophoresis in a number of capillary columns, however, it is difficult to recognize which signal corresponds to each capillary column. Furthermore, it is also difficult to recognize which bases are currently detected in the respective capillary columns. Consequently, it is difficult to grasp the electrophoresis states.